In the past decade, mitochondrial fusion and fission have emerged as major regulators of mitochondrial function. Defects in these processes are associated with human disease, and mouse studies indicate their importance in maintaining a healthy mitochondrial population within cells. This proposal focuses on the mechanism of mitochondrial fission, which is implicated in control of mitochondrial morphology, degradation of mitochondria by autophagy, control of mitochondrial distribution, and regulation of apoptosis. During the process of mitochondrial fission, Drp1 (dynamin-related protein 1) is recruited from the cytosol onto the mitochondrial surface. Here it assembles into an oligomeric complex that wraps around the mitochondrial tubule and constricts it to mediate membrane scission. In order for Drp1 to be recruited to mitochondria, the mitochondrial outer membrane contains four Drp1 receptors: Fis1, Mff, MiD49, and MiD51. Cellular studies indicate that the latter three molecules are the most functionally important for mitochondrial fission. This proposal focuses on obtaining a structural, biochemical, and cellular understanding of how these molecules mediate Drp1 recruitment. In Aim 1, X-ray crystallography is used to obtain atomic structures of Mff, MiD49, and MiD51. Current work has already yielded a high-resolution structure of MiD51. Surprisingly, this structure indicates that MiD51 has a nucleotidyl transferase domain, with conservation of key residues that are implicated in nucleotide binding and catalysis. Additional structures of MiD51 in complex with nucleotide will be obtained. In Aim 2, biochemical studies are used to advance the understanding of MiD51 and MiD49 function. Given that MiD51 has a nucleotidyl transferase fold, the nucleotide binding specificity of MiD51 will be measured with quantitative binding assays. In addition, enzymatic assays will be used to determine if MiD51 has a catalytic function in nucleotide transfer. Similar studies will be done with MiD49, which is homologous to MiD51. In Aim 3, structure-function analysis in cell culture is used to determine whether nucleotide binding, catalysis, or dimerization is important for the ability of MiD51 to recruit Drp and mediate fission. MiD51 and MiD49 knockout cells for further structure-function analysis will be generated through the use of genome editing technology. Finally, a genetic approach will be utilized to map the Drp1 binding site on Mff, MiD49, and MiD51. Taken together, these studies will greatly advance the understanding of the structural biology of Drp1 receptors and may ultimately lead to new methods to modulate mitochondrial fission. 1